Li Xian-Wei, Zuo Dong-Ze, Shen Yuan-Yuan, Hao Wei
Department of Pharmacology, Wannan Medical College, Wuhu 241002, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2017 Jan 8;33(1):16-21. doi: 10.12047/j.cjap.5448.2017.004.
To observe the effects of calcitonin gene-related peptide (CGRP) on eukaryotic translation initiation factor 3a (eIF3a) and p27 expression in bleomycin-induced pulmonary fibrosis of rats and its possible mechanism.
Twenty-four male SD rats weighing 180~220 g were randomly divided into three groups (=8):control group, bleomycin group, bleomycin plus capsaicin group. Bleomycin (5 mg/kg) was used to induce pulmonary fibrosis rat model. Rats were given capsaicin (50 mg/kg·d) by subcutaneous injections 4 days before to deplete endogenous CGRP. At the end of experiments, blood samples were collected from carotid artery to determinate the plasma levels of CGRP by ELISA. The cells were divided into 6 groups as follows:control group, transforming growth factor-β1 (TGF-β1) group, +CGRP (1, 10, 100 nmol/L) group, +CGRP 100 nmol/L and CGRP8-37 1 mol/L group respectively(=9). TGF-β1 (5 ng/ml) stimulated proliferation of pulmonary fibroblasts and proliferation was measured by BrdU marking. The expression levels of eIF3a, p27, α-smooth muscle actin (α-SMA) and collagen Ⅰ were detected by immunohistochemisty, real-time PCR or Western blot.
The expressions of eIF3a, α-SMA, and collagen I were increased and the expression of p27 was decreasing in pulmonary fibrosis rats induced by bleomycin. Exogenous application of CGRP significantly inhibited TGF-β1-induced proliferation and differentiation of pulmonary fibroblasts and the expressions of α-SMA, collagen I and eIF3a, and upregulated the expression of p27. All these effects of CGRP were abolished in the presence of CGRP8-37.
These results suggest that endogenous CGRP is related to the development of pulmonary fibrosis induced by bleomycin, and the inhibitory effect of CGRP on proliferation of lung fibroblasts involves the eIF3a/p27 signaling pathway.
观察降钙素基因相关肽（CGRP）对博来霉素诱导的大鼠肺纤维化中真核翻译起始因子3a（eIF3a）和p27表达的影响及其可能机制。
将24只体重180~220 g的雄性SD大鼠随机分为3组（每组8只）：对照组、博来霉素组、博来霉素加辣椒素组。采用博来霉素（5 mg/kg）诱导大鼠肺纤维化模型。在造模前4天，大鼠皮下注射辣椒素（50 mg/kg·d）以耗尽内源性CGRP。实验结束时，从颈动脉采集血样，用酶联免疫吸附测定法（ELISA）测定血浆CGRP水平。细胞分为以下6组：对照组、转化生长因子-β1（TGF-β1）组、+CGRP（1、10、100 nmol/L）组、+CGRP 100 nmol/L和CGRP8-37 1 μmol/L组（每组9个样本）。用TGF-β1（5 ng/ml）刺激肺成纤维细胞增殖，通过BrdU标记法检测增殖情况。采用免疫组织化学、实时荧光定量聚合酶链反应（real-time PCR）或蛋白质免疫印迹法（Western blot）检测eIF3a、p27、α-平滑肌肌动蛋白（α-SMA）和Ⅰ型胶原的表达水平。
在博来霉素诱导的肺纤维化大鼠中，eIF3a、α-SMA和Ⅰ型胶原的表达增加，p27的表达降低。外源性应用CGRP可显著抑制TGF-β1诱导的肺成纤维细胞增殖和分化以及α-SMA、Ⅰ型胶原和eIF3a的表达，并上调p27的表达。在CGRP8-37存在的情况下，CGRP的所有这些作用均被消除。
这些结果表明内源性CGRP与博来霉素诱导的肺纤维化的发生发展有关，CGRP对肺成纤维细胞增殖的抑制作用涉及eIF3a/p27信号通路。
